On 13 Apr 2001 00:12:37 +0100,
TFitzwater at Gilead.com (TFitzwater at Gilead.com) wrote:
>>Daniel Mebrahtu (danielmeb at hotmail.com)
>>Wed 11 Apr 2001 - 00:16:32 BST
>>dear members,
>>i am having a problem ligating Pvu I ends in a very important project i
>>started recently. can anyone help me out if she/he have had a similar
>>problem and triumphed in the end.
>>daniel
>> I have seen this problem before (many years ago), and later ran across a
> reference that the Pvu I ends need to come from a dam minus strain of E.
> coli if you want to ligate these ends. You can cut dam plus DNA with Pvu
> I, but you can't religate it. This is not something the restriction enzyme
> suppliers mention. Both the vector and the insert need to be isolated from
> dam minus E. coli. (The reference is at home, so I can't tell you what it
> was right now.)
It is true that PvuI-digested DNA isolated from dam(-) strains ligates
more efficiently than DNA isolated from dam(+) strains -- the NEB catalog
documents it, and we too have observed it.
However this difference in ligation efficiency may be dependent upon the
sequences immediately juxtaposed to the PvuI site. In our experience
ligation efficiency of the PvuI site in the beta-lactamase gene is
apparently independent of the Dam methylation status of the plasmid.
Unlike your experience, after ligation the PvuI site was not destroyed;
however, because it is in the marker being selected for, perhaps deletions
are selected against.
Later,
Ashok
--
Ashok Aiyar, Ph.D.
Assistant Professor email: a-aiyar at northwestern.edu
Department of Microbiology-Immunology office: (312) 503-2524
303 E. Chicago Avenue, WARD 4-123 lab: (312) 503-2542
Northwestern University, Chicago, IL 60611 fax: (312) 503-1339